BSc - MSc (Int.)

The present research work entitled ‘Characterization of virus(es)
involved in Chilli leaf curl disease complex in Chilli (Capsicum annuum L.)’ was carried out at Department of Plant Biotechnology and Department of Plant
Pathology, College of Agriculture, Vellayani, during the year 2020-2021, with
the primary objective to characterize the virus(es) causing Chilli leaf curl
disease from five agro-ecological zones of Kerala. Symptomatic chilli plants were collected from different chilli growing
agro-ecological zones (AEZs) of the state viz., Nedumangad from
Thiruvananthapuram (coastal plains), Kumarakom from Kottayam (Central
midlands), Vellanikkara from Thrissur (South Midlands), Thekkepotta from
Palakkad (Palakkad plains) and Meenangadi from Wayanad (High Ranges). Highest disease incidence of 80 per cent was observed in Thiruvananthapuram. Symptomatological studies revealed that the infected plants showed
extensive curling, crinkling and puckering, apical bunchy appearance, slight
yellowing with mosaic patterns on the leaf lamina. Inter-veinal chlorosis
accompanied with necrosis and vein-banding was also observed. The
association of white- flies (Bemisia tabaci) on the adaxial surfaces of leaves was
the best indication for viral infection in chilli plants. Genomic DNA isolated from infected chilli leaf samples using CTAB
method showed an absorbance value of 1.8 and genomic RNA extracted using
TRIzol method gave an absorbance value of 2.0.
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Partial characterization of coat protein gene of DNA and RNA viruses
were carried out using coat protein specific primers. It produced amplicons of ~550bp specific to AV-AC primers from Thiruvananthapuram, Thrissur and
Palakkad and ~500bp amplicons specific to Deng primers from all the AEZs
under study. Synthesized cDNA was amplified using the reported coat-protein
specific primers viz., CMV and CVMV primers. ~550bp sized amplicons were
obtained specific to CMV primers from all the zones except Wayanad where
the amplicons obtained were of size ~700bp specific to CVMV primers. Eluted amplicons were ligated to pMD20-T vector, cloned to
competent DH5α E.coli cells and screened through Blue- white screening on
LB ampicillin/X-Gal plate wherein recombinant transformed cells appeared as
white colonies. Those transformed colonies that gave an expected band size in
Colony PCR were chosen for sequencing. Sequenced coat protein gene of DNA virus isolated from
Thiruvananthapuram, Kottayam, Thrissur, Palakkad and Wayanad resulted in
sequence lengths of 558bp, 512bp, 560bp, 515bp and 520bp respectively while
that of RNA viruses yielded sequences of length 560bp, 556bp, 558bp, 563bp
and 690bp respectively. BLASTn analysis of the DNA viral sequences revealed that
Thiruvananthapuram (OM719034), Kottayam (OM719035), Thrissur
(OM719036), Palakkad (OM719037) and Wayanad (OM719038) had 98 per
cent, 98 per cent, 97 per cent, 98 per cent and 89 per cent similarity with
Tomato leaf curl New Delhi Virus. RNA isolates from Thiruvananthapuram
(OM719039), Kottayam (OM719040), Thrissur (OM719041) and Palakkad
(OM719042) showed an identity of 99.46 per cent, 99.09 per cent, 98.54 per
cent and 99.46 per cent with Cucumber mosaic virus isolates and that from
Wayanad (OM719043) showed 97 per cent similarity with Chilli veinal mottle
virus.
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Phylogenetic relationship among the DNA isolates showed maximum
relatedness between Thiruvananthapuram and Thrissur followed by Kottayam
and Palakkad and the Wayanad isolate was categorized as an out group. RNA
isolates revealed maximum relatedness between Thiruvananthapuram and
Kottayam while that from Palakkad was categorized as out group. To conclude, partial characterization of viruses isolated from chilli
plants selected for the study revealed that the chilli plants collected for the
study were subjected to mixed infections caused by a complex of viruses. The
plants from Thiruvananthapuram, Kottayam, Thrissur and Palakkad were
infected by a complex of viruses that belong to genera Begomovirus and
Cucumovirus while those plants from Wayanad were infected by a complex
comprising of viruses of genera Begomovirus and Potyvirus.